Droplet generation

ABSTRACT

Perfectly spherical, smooth and uniform microcapsules, which may contain living cells, are produced having a diameter less than 700 μm by employing an electrostatic droplet generator. A droplet is suspended from a pointed source, such as a needle, and is charged with high static voltage. A collecting vessel or ring device is charged with opposing polarity and attracts the droplet. When a voltage potential threshold is passed, the droplet moves from the source to the collecting vessel. The voltage pulse height, pulse frequency and length, and extrusion rate of the droplets are adjustable so that predetermined sizes of droplets may be repeatedly generated and collected.

This is a division of Ser. No. 631,971, filed 7/16/89 now U.S. Pat. No.4,789,550.

FIELD OF THE INVENTION

The present invention is concerned with droplet generation, particularlywith respect to droplet generation in the encapsulation of living cellsor individual cells in microcapsules.

BACKGROUND TO THE INVENTION

Various attempts have been made over the past twenty years to providesemi-permeable microcapsules which were both biocompatible with the bodytissue and impermeable to the components of the immune system. Typicalof such attempts is that described in U.S. Pat. Nos. 4,352,883 and4,391,909 to Franklin Lim.

As set forth therein, living tissue or individual cells are suspended inan aqueous solution of a reversibly-gellable material, typically sodiumalginate, and droplets of this suspension are allowed to drop into ahardening solution, typically calcium chloride. The temporary capsulesso formed are then treated with polylysine and polyethyleneimine to forman outer semi-permeable coating. The core material is reliquified byion-exchange of the calcium ions.

Survival times of microcapsules produced by this prior art procedure inthe animal body were consistently less than 3 weeks, thereby severelylimiting the utility of this prior art encapsulation procedure in thetreatment of diseases requiring organ transplantation, such as diabetesand liver disease.

In copending U.S. Pat. application Ser. No. 501,445 filed June 6, 1983,assigned to the assignees hereof, the disclosure of which isincorporated herein by reference, there is described an improvement onthe above-mentioned prior art procedure which forms a semi-permeablemembrane which is both biocompatible and yet is able to protecttransplanted tissue and cells from destruction by the immune system,such that, in animal tests, a single intraperitoneal transplant ofencapsulated islets reversed the diabetic state for more than one year.

The success of the procedure according to the aforesaid applicationresults from a semi-permeable and durable membrane which has an outersurface of biocompatible negatively-charged material. The improveddurability, i.e. resistance to rupture, of these microcapsules is due totheir near perfect spherical shape and enhanced capsule membranethickness.

Although the microcapsules produced in the aforesaid pending applicationrepresent a significant advance in the treatment of diseases requiringorgan transplantation, there is one drawback which inhibits. more idealutilization of the microcapsules and this drawback arises from therelatively large size of the individual microcapsules, which have adiameter from 700 to 1000 μm. Microcapsules produced according to theprocedure of the Lim patents also had relatively large diameters ofabout 1000 to 2000 μm. Microcapsules having these diameters cannot beinjected directly into the cardiovascular system, since they wouldocclude the blood vessel. Accordingly, the microcapsules must beimplanted into large body cavities, such as the intraperitoneal cavity.

Location of the implants in an area of the body other than thecardiovascular system results in an increase in the response time of themicrocapsules to changing blood conditions, since the microcapsules arenot directly in contact with the blood stream. In addition, therelatively large size of the microcapsules compared to themicroencapsulated tissue or cells (e.g. about 200 μm for islets ofLangerhans) results in a high diffusional resistance for moleculespassing through the microcapsule core.

An air jet-syringe pump extrusion method was used in the procedure ofthe aforementioned pending application and in the Lim patents to productgel droplets containing entrapped islets, or other tissue or cells, fromthe suspension of the islets in aqueous sodium alginate solution. Inthis procedure, the sodium alginate solution is extruded through aneedle located inside a sheathed tube through which air flows at acontrolled rate. As liquid droplets are forced out of the end of theneedle by the syringe pump, the droplets are pulled off by the shearforces set up by the rapidly-flowing air stream. The higher thevolumetric air flow rate, the stronger are the shear forces, the morequickly the droplets are pulled off the end of the needle and thesmaller are the resultant droplets.

However, there are inherent restraints in this prior art procedure whichprevent the size of microcapsule produced thereby being less than 700microns. These restraints are that the viscosity of the gel-formingliquid must be greater than 30 cps in order to form perfectly sphericalcapsules, the minimum internal diameter of the needle must be greaterthan 300 μm (24 gauge) so as to prevent blockage of the needle by theislets, and the volumetric air flow rate must remain below 2000 cc/minin order to produce capsules of uniform diameter.

SUMMARY OF INVENTION

We have now discovered an improved procedure and apparatus for formingperfectly spherical, smooth and uniform droplets, such that there can beproduced therefrom perfectly spherical, smooth and uniform microcapsuleshaving a diameter of less than 700 μm, preferably about 150 to about 500μm. Such microcapsules constitute one aspect of the present invention.

The novel microcapsules are formed of biocompatible material and containliving tissue or cells as a core material. A preferred core material isislets of Langerhans, so as to effect long term control of blood sugarlevels in diabetic animals, including humans, by cardiovascularinjection of biocompatible microencapsulated islets of Langerhans.

The provision of smaller diameter microcapsules in accordance with thisinvention, for example 200 to 300 μm, permits direct injection of themicrocapsules into the blood stream, so that they may eventually lodgeinside body organs, such as the liver or spleen, where they arecontinuously washed with fresh blood. The direct contact between themicrocapsule and the blood significantly decreases the response time ofthe encapsulated tissue or cells to any biochemical change and therebyincreases its efficiency. In addition, the smaller microcapsules resultin a lower diffusional resistance for molecules passing through themicrocapsule core, further increasing the efficiency of the cells. As aresult, fewer of the smaller diameter microencapsulated islets ofLangerhans need to be transplanted per kilogram of recipient body weightto achieve prolonged control of blood sugar levels in diabetic patients.

The living tissue-containing microcapsules also may be injected orimplanted into any other convenient location in the body to be treatedthereby, although this manner of administration is less preferred forthe reasons noted above.

The small diameter microcapsules provided in accordance with thisinvention are formed by employing an electrostatic droplet generator inthe initial gel-droplet-forming step. In this procedure, whichconstitutes a second aspect of the invention, a droplet, suspended froma source, is charged with a high static voltage and a second location,for example, a collecting vessel, is charged with opposite polarity, soas to attract the droplet. When a threshold of voltage differencebetween the locations is passed, the droplet moves from the sourcetowards the second location and, thereby, to the collecting vessel.

An adjustable high voltage pulse is generated and applied to the dropletformed on the end of a needle by a syringe pump. The height of thevoltage pulse, the pulse frequency and the pulse length are synchronizedwith the amount of material dispensed, so that known sizes of dropletscan be repeatedly generated and collected.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic representation of a droplet-forming generatorconstructed in accordance with one embodiment of the invention;

FIG. 2 is a schematic representation of a droplet-forming generator,constructed in accordance with a second embodiment of the invention;

FIG. 3 is a schematic block diagram representation of the circuitryrequired to apply electricity to the droplet generator of FIG. 1 or 2;and

FIG. 4 is a graphical representation of a typical waveform of the outputof the droplet generator circuitry of FIG. 3.

DESCRIPTION OF PREFERRED EMBODIMENTS

Referring to the drawings, FIG. 1 shows a droplet-forming apparatus 10constructed in accordance with one embodiment of the invention. As showntherein, a syringe 12 of non-conducting, usually polymeric, materialcontains a gel droplet-forming liquid 14 which contains living cells. Aplunger 16 is driven by a syringe pump 18 to expel droplets 20 from thelower end of a stainless steel syringe needle 22 communicating with thelower end of the syringe reservoir 12, towards a collecting vessel 24containing a hardening solution 26, which may be aqueous calciumchloride solution in the case of an aqueous droplet-forming liquidcontaining sodium alginate.

The positive lead 28 of an electrical pulse generator (see FIG. 3) isconnected to the needle 22 while the negative lead of the pulsegenerator is connected to the hardening solution.

The needle 22 may be bevelled at its outlet tip 27, if desired. The tip27 is located at a specific distance from the top of the recipientmedium 26 in the collecting vessel 24 consistent with the voltage pulseto be applied therebetween to effect droplet formation. The size of thedroplets 18 may be varied by varying the distance between the needle tip27 and the liquid in the collecting vessel 24, with shorter distancesleading to smaller droplets, by varying the voltage applied by the leads28 and 30 with increased voltage leading to smaller droplets, by varyingthe pulse length of applied electricity with decreasing pulse lengthleading to smaller droplets, or by varying the speed of the pump 18 withdecreasing pump speed leading to smaller droplets.

FIG. 2 illustrates an alternative arrangement wherein the positive wire28 from the pulse generator is detached from the needle 22 and insteadis attached to a stainless steel ring 32 which is mounted to the lowerend of a conical support 34 of non-conductive material which surroundsand extends below the needle 22. The negative lead 30 is attached to theneedle 22 rather than to the recipient medium 26. In this arrangement,the distance between the tip 27 of the needle 22 and the top of therecipient medium 26 does not affect the gel droplet size.

The static voltage which is applied by lead wires 28 and 30 duringdroplet formation in FIGS. 1 and 2 results in gel droplets having adiameter less than about 700 μm, preferably about 150 to about 500 μm.These droplets then may be coated with a thin coating of asemi-permeable biocompatible membrane. The resulting microcapsules aresmall enough to be injected into an animal body using an 18 gauge needlefitted to a syringe.

Since the voltage applied during droplet formation is a static one, theviability of encapsulated living tissue, such as islets of Langerhans orliver cells, is not destroyed, and hence the microencapsulated livingtissue is capable of on-going metabolism.

Referring now to FIGS. 3 and 4 of the drawings, an electrostatic pulsegenerator 110 suitable for formation of electrostatic pulses to beapplied during droplet formation by the apparatus of FIG. 1 or 2 isillustrated in FIG. 3. As seen therein, the pulse generator 110 includesan isolated power supply 112, which may be connected to any desiredsource of electric power, logic circuitry 114, console panel 116 havingadjusting knobs for pulse frequency 118, pulse width 120 and highvoltage output 122, a pulse amplifier 124, and a high voltagetransformer and rectifier 126 which outputs to the electrical lead wires28 and 30.

The electrical pulse voltage, pulse frequency and pulse length whichpass to the droplet forming apparatus 10 by the lead wires 28 and 30 mayeach vary widely, depending on the size of droplets desired. The pulsevoltage, which determines the strength of the force pulling the dropletsfrom the end of the needle 22, usually varies from about 1 to about 25KV. The pulse frequency, which determines how many pulses are applied tothe droplet, usually varies from about 10 to about 100 sec⁻¹. The pulselength, which determines the length of time for which thedroplet-forming force is applied, usually varies from about 1 to about 6m. sec. The interaction of the various time periods and their meaning isfurther illustrated in FIG. 4. These values are synchronized with theamount of material dispensed from the needle to obtain uniformly-sizeddroplets.

These specific design parameters ensure that there is no voltage overlapsince a pulse lasts for 1 to 6 m. sec with each pulse occurring every 10to 100 m. sec. Accordingly, a minimum of 4 m. sec and a maximum of 99 m.sec occurs between pulses. There is, in addition, a low baseline voltagewhich maintains the forming droplet in position between pulses.

There are numerous examples in the prior art of devices forelectrostatically sorting biological cells, electrostatic sprays fordispensing paints and/or polymers and electrostatic droplet generatorsfor ink printing. Illustrative examples of such devices are described inU.S. Pat. Nos. 4,347,935, 4,395,716 and 4,097,373 and British Pat. No.1,346,301. The droplet generators described in these prior patents usean external excitation source, such as acoustic vibration, for theinitial formation of the droplet. The droplets are chargedelectrostatically only after they leave the generator, while in thepresent invention, the droplet-forming liquid 14 is charged directly byhigh static voltage. In the prior art arrangements, the externalvibrating source causes formation of the droplets while in the presentinvention droplets are produced by direct electrostatic interaction.

The droplet generator of this invention is capable of producing verysmall, spherical droplets containing living cells with each step of thedroplet formation being under the direct control of the operator.

Although the disclosure herein is directed mainly to the encapsulationof living tissue or cells for the specific purposes and advantagesoutlined above, it will be understood that the electrostatic dropletforming method and apparatus described herein has application in otherfields and, for example, may be used in spray painting and ink printing.

GENERAL DESCRIPTION OF MICROENCAPSULATION

In this invention, living tissue or individual cells are encapsulated ina biocompatible semi-permeable membrane, in the form of a hydrogel. Thematerial to be encapsulated is suspended in a physiologically-compatiblemedium containing a water soluble substance which can be reversiblygelled to provide a temporary protective environment for the tissue. Themedium is formed into droplets containing the tissue, using the dropletgeneration procedure of the invention, and gelled, for example, bychanging conditions of temperature, pH or ionic environment, to formtemporary capsules, of substantially perfect spherical shape.Thereafter, the temporary capsules which result are treated to form amembrane of controlled permeability and negatively-charged outer surfaceabout the shape-retaining temporary capsules. The semi-permeable natureof the membrane permits nutrients and oxygen to low to the core materialand metabolic products to flow therefrom while retaining the corematerial within the microcapsule. The biocompatible nature of thesemi-permeable membrane allows the passage of such materials to and fromthe core to occur without inflammation or other adverse body responsewhile the outer negatively-charged surface inhibits surficial cellgrowth, so that the membrane remains semi-permeable and effective forextended periods of time, typically from three to six months or longer.

The temporary capsules may be formed from any non-toxic water-solublesubstance that can be gelled to form a shape retaining mass by a changeof conditions in the medium in which it is placed, and also comprisesplural groups that are readily ionized to form anionic or cationicgroups. The presence of such groups enables surface layers of thecapsule to cross-link to produce a permanent membrane when exposed topolymers containing multiple functionalities of the opposite charge.

Preferably, the temporary capsules are formed from a polysaccharide gum,which may be natural or synthetic, of a type that can be gelled to forma shape retaining mass by exposure to a change in conditions and can bepermanently cross-linked or hardened by polymers containing reactivegroups, such as amino groups, which can react with the acidicpolysaccharide constituents. Most preferably, the gum is alkali metalalginate, specifically sodium alginate, although other water-solublegums may be used.

The temporary capsules may be formed from sodium alginate by extrudingdroplets of aqueous sodium alginate solution into an aqueous calciumchloride solution. It is preferred that the temporary capsules besubstantially spherical so that perfectly spherical microcapsules can beformed for cardiovascular injection. Substantially perfectly sphericaltemporary capsules are formed by using an aqueous sodium alginatesolution having a viscosity of at least about 30 centipoise. Atviscosities below this critical lower limit, the temporary capsules havean irregular shape. Perfectly spherical capsules are obtained over awide range of viscosity of the sodium alginate solution above thecritical lower limit of 30 centipoise, with an upper limit beingdictated largely by the ability to extrude the solution into thehardening medium. However, it has also been found that the minimum sizeof perfectly spherical droplet which can be obtained at a viscosity ofat least about 30 cps increases with increasing viscosity.

Formation of the permanent semi-permeable membrane about the temporarycapsules preferably is effected by ionic reaction between free acidgroups in the surface layer of the gelled gum and biocompatible polymerscontaining acid-reactive groups, such as, amino groups, typically in adilute aqueous solution of the selected polymer.

The cross-linking biocompatible polymers which may be used includepolyamino acids, preferably polylysine. It is noted thatpolyethyleneimine and other imine-containing polymers are unsuitable formembrane formation in view of their non-biocompatible nature. Themolecular weight of the preferred polylysine polymer should becontrolled within a narrow range of about 10,000 to about 30,000,preferably about 17,000, to achieve the required membrane porosity. Theuse of polylysine or other polyamino acid results in microcapsuleshaving a positively-charged surface, which would be unsuitable for longterm viability, although the microcapsules are biocompatible. It isimportant for long term in vivo life for the polylysine or otherpolyamino acid to be reacted for a period of time sufficient to developa substantial thickness of membrane, so as to provide a substantialnumber of surface groups for post-reaction, as discussed below,sufficient structural strength to permit in vivo injection andsufficient quantity of biocompatible polymer to permit in vivostructural integrity to be retained. Usually, for polylysine of themolecular weight range noted above, a reaction time of at least sixminutes is required to achieve these results, preferably at least aboutnine minutes, generally up to about 9 minutes. These reaction timesresult in a polylysine layer thickness of about 5 microns.

Surprisingly, the actual strength of the aqueous solution of polylysineused to react with the temporary. capsules does not affect the capsulewall thickness, at concentration levels in excess of about 0.05 wt. %.

The semi-permeable membrane formed about the temporary capsules by thereaction with the polyamino acid next is treated with a non-toxicbiocompatible water-soluble polymeric material which is capable of ionicreaction with free amino groups to form an outer negatively-chargedcoating about the membrane, typically by suspension of the microcapsulesin an aqueous solution of the polymeric material. The material used toform the outer coating preferably is the same material as is used toform the temporary capsules, preferably a polysaccharide gum, morepreferably an alkali metal alginate, such as, sodium alginate. Otherbiocompatible polymeric materials containing base-reactive groups, suchas, polyvinyl alcohol and poly beta-hydroxy butyric acid, may be used toform the outer coating to the microcapsules. Molecular weights of suchpolymeric materials typically vary from about 10⁴ to about 10⁶.

The biocompatible water-soluble polymeric material containingamino-reactive groups reacts with the outer amino-groups of thesemi-permeable membrane to form an outer coating. Thus outer coatingpermanently shrouds the polyamino acid layer, although leaving intactthe porosity of the semi-permeable membrane, and provides anegatively-charged surface. By virtue of the number of surface aminogroups on the polyamino acid membrane, resulting from the prolongedreaction time, the outer negatively-charged polymer coating resistsdegradation and removal, in vivo, so that the positively chargedsurfaces are not exposed to the body environment.

The treatment of the polyamino microcapsules with the biocompatiblebase-reactive material retains the overall biocompatible nature of thesemi-permeable membrane and results in a negatively-charged outersurface which inhibits cell growth and, therefore, permits thesemi-permeable membrane to retain its permeability and henceeffectiveness over an extended period of time.

Following formation of the microcapsules, reliquification of thesuspending medium for the core material may be effected byre-establishing the conditions under which the material is liquid. Thismay be achieved by ion exchange to remove multivalent cation, forexample, by immersion in phosphate buffered saline or citrate buffer.The reliquification step, though beneficial in decreasing diffusionresistance, is not essential for the provision of an effective productand may be omitted, since it has been shown that transplanted islets(rat to mouse) in microcapsules whose interiors have not beenreliquified, are also effective in normalizing blood sugar levels ofdiabetic animals. Surprisingly, the calcium alginate gel core does notreliquify inside the body, since intact gel cores have been found inmicrocapsules recovered from diabetic animals up to one year afterimplantation.

The process of the invention may be used to encapsulate living tissue,multicellular fractions thereof or individual cells, for example, isletsof Langerhans, liver cells and red blood cells, and otherbiologically-active material. The microcapsules which result may beimplanted into an appropriate site within a mammalian body for thepurpose of providing the body with the specialized physiologicalfunction of the tissue while the tissue remains viable. The implantationmay be achieved by simple injection, so that surgical procedures are notrequired. As noted earlier, cardiovascular injection may be effected, inview of the smaller diameter microcapsules which result from theelectrostatic droplet generation procedure.

The core of the microcapsules contains the living tissue cells and anaqueous medium of nutrients sufficient to maintain the tissue and allowits normal metabolism. The cells are viable, physiologically active andcapable of ongoing metabolism.

The biocompatible semi-permeable membrane encapsulating the corematerial consists of interpenetrating layers of ionically-interactedbiocompatible materials. The overall wall thickness of thesemi-permeable membrane usually varies from about 4 to about 6 μm. Themicrocapsules themselves have a diameter in the range of less than about700 μm, preferably in the range of about 150 to about 500 μm formicrocapsules containing islets of Langerhans as the core material. Thebiocompatible semi-permeable membrane is in the form of a hydrogel andhence has an overall water content within the membrane structure of atleast about 20 wt %, which may vary up to about 95 wt %, depending onthe molecular weight of the polyamino acid.

In a particularly preferred embodiment of the invention, living cellsare microencapsulated within a polylysine-alginate semi-permeablehydrogel. The cells are initially suspended uniformly in a sodiumalginate solution in physiological saline. Where the microcapsules areto be used for the treatment of diabetes by controlling blood sugar inanimals, including humans, the living cells take the form of islets ofLangerhans from an animal pancreas.

Spherical droplets containing the cells are produced from an aqueoussodium alginate solution by the electrostatic droplet generationprocedure of the invention and are collected as gelled spheres in ahardening solution, such as, calcium chloride. The gelled spheres arecoated with polylysine followed by an outer coating of sodium alginateThe microcapsules may then be suspended in isotonic sodium citrate orother convenient ion exchange medium to reliquify the alginate gelinside the microcapsule to restore the cells to a mobile state. As notedearlier, this step may be omitted, if desired.

The outer biochemically inert but biocompatible alginate surface is anegatively-charged hydrogel containing up to about 95% water. The lowinterfacial tension between the swollen gel surface and the aqueousbiological environment minimizes protein interaction, otherwise a strongprotein-polymer interaction may cause a severe inflammatory response.The biocompatibility of the hydrogel membrane leads to long termviability of the capsules when implanted. Polyethyleneimine-surfacedmicrocapsules do not appear to possess this property, since they producea strong inflammatory response and hence are rejected by the body, whichseverely limits the useful in vivo life of the microcapsules. The softrubbery consistency of most hydrogels may also contribute to theirbiocompatibility by decreasing frictional irritation to surroundingtissues.

The strength of the microcapsules may be increased by additionalcross-linking, for example, using glutaraldehyde, prior toreliquification of the gel, if effected.

For in vivo implantation, it is not essential that the biocompatibleouter surface be composed of sodium alginate, but it is essential thatthe outer surface be biocompatible and negatively-charged. Bindingoccurs between the negatively-charged groups, usually hydroxyl orcarboxyl groups, of the biocompatible outer surface material, and thepositively-charged amino groups on polylysine.

By the present invention, therefore, there have been obtainedbiocompatible microcapsules capable of long term in vivo life and havinga diameter which render them suitable for injection of living tissueinto the blood stream, so that the microcapsules may lodge inside bodyorgans for ongoing metabolism therein. While the primary benefit of thesmaller diameter microcapsules of the invention is in in-vivo uses, theliving tissue-containing microcapsules may also be put to a variety ofin-vitro uses.

In addition to producing microcapsules containing living tissue orcells, the present invention may be used to form microcapsulescontaining a variety of other core materials, depending on the intendedend use of the microcapsules.

EXAMPLES Example 1

This Example illustrates the formation of small diameter gel dropletsusing an electrostatic droplet generator.

An apparatus as illustrated in FIG. 1 was set up. A 1.5% w/v sodiumalginate solution (14) was placed in a 10 cc syringe (12) to which isattached a 22 gauge stainless steel needle (22) having a 90° beveloutlet. The positive polarity wire (28) was attached to the metal leurlock section of the needle and the needle-syringe combination wasattached to the syringe pump (18). A 1.1% calcium chloride solution (26)was poured into a 4"×1" petri dish (24) to which was attached thenegative polarity wire (30). The petri dish (24) was positioned so thatthe liquid surface therein was 10 mm from the tip of the needle (22).

The pulse voltage dial (122) on the adjustment panel (116) was set at 12KV, the pulse frequency dial (118) at 20 sec⁻¹, the pulse length dial(120) at 2 m. sec, and the syringe pump speed at 4 ml/hr. The syringepump (18) and droplet generator were both turned on so that sodiumalginate liquid droplets (20) were drawn from the tip (27) of the needle(22) and, upon entering the calcium chloride solution in the petri dish(24), calcium alginate gel droplets were formed and were collectedtherein. The resultant calcium alginate gel droplets were found to beperfectly smooth and spherical and with a mean diameter of 300 (±50 SD)μm.

The syringe needle (22) used in this Example was of the same diameter aswas previously used in an air jet syringe wherein a rapidly flowing airstream was used to remove sodium alginate liquid droplets from the tip(27) of the needle (22) using the air jet syringe, the smallest diametercalcium alginate gel droplets attainable had a diameter of 700 μm. Theelectrostatic procedure described in this Example, therefore, was ableto decrease the gel droplet diameter to approximately half this value.

Example 2

This Example illustrates the formation of small diameter gel dropletsusing an alternative form of droplet generation.

The procedure of Example 1 was repeated, except that the apparatus ofFIG. 2 was utilized, i.e. the negative polarity wire (30) was attachedto the needle (22) and the positive polarity wire (28) is attached tothe metal ring device (32) which is spaced downwardly from the tip ofthe needle (22). The centre of the metal ring (32) was positioned 7 mmdownwardly from the tip (27) of the needle (22) and an uncharged petridish (24) was positioned about 5 cm downwardly from the ring assembly.

The calcium alginate gel droplets produced by this procedure andcollected in the petri dish were observed to be perfectly smooth andspherical and to have a mean diameter of 450 (±65 SD) μm. When theexperiment was repeated with the charge reversed, a greater variation ofgel droplet diameter was observed with the standard deviation (SD) ofgel droplet diameter approximately doubling.

Example 3

This Example illustrates the viability of living tissue after passagethrough the electrostatic droplet generator.

The procedure of Example 1 was repeated except that islets of Langerhansextracted from the pancreatic tissue of dogs were added to the sodiumalginate solution in the syringe in a concentration of 500 islets/2 mland the calcium chloride solution was replaced by saline, so that geldroplet formation did not occur in this experiment. After passagethrough the electrostatic droplet generator, 100% of the islets wereshown to be viable using Trypan blue staining. All the islets appearedwhite when viewed under the microscope, there being no evidence of theblue appearance characteristic of dead islets.

Example 4

This Example illustrates the formation of small semi-permeablemicrocapsules containing islets of Langerhans.

Cultured at islets of Langerhans (2×10³ islets in 0.2 ml medium) weresuspended uniformly in 2 ml of a 1.5% (2/2) sodium alginate solution(viscosity 51 cps) in physiological saline. Spherical dropletscontaining islets were produced with an electrostatic droplet generatorusing the procedure of Example 1 and were collected in 1.5% (w/w)calcium chloride solution. The supernatant was decanted and the gelledspherical calcium alginate droplets, containing islets, were washed withCHES (2-cyclohexylaminoethane sulfonic acid) solution and 1.1% calciumchloride solution.

After aspirating off the supernatant, the gelled droplets were incubatedfor 6 minutes in 0.05% (w/w) solution of polylysine having a molecularweight of 17,000. The supernatant was decanted and the polylysinecapsules were washed with dilute CHES, 1.1% calcium chloride solutionand physiological saline.

The washed polylysine capsules were incubated for 4 minutes in 30 ml of0.03% sodium alginate to permit the formation of an outer alginatemembrane on the initial polylysine membrane, by ionic interactionbetween the negatively-charged alginate and the positively-chargedpolylysine.

The resulting microcapsules were washed with saline, 0.05 M citratebuffer for 6 minutes to reliquify the inner calcium alginate, and afinal saline wash. The microcapsules were found to be perfectlyspherical and each to contain from 1 to 2 viable islets. Themicrocapsules had a mean diameter of 300 (±50 SD) microns and wallthicknesses of 5 μm. The microapsules were suspended in nutrient mediumat 37° C.

The viability of the islets was demonstrated by Trypan Blue stainingafter the capsule walls were dissociated with heparin.

Example 5

This Example illustrates the formation of small semi-permeablemicrocapsules containing hepatocytes (liver cells).

The procedure of Example 4 was repeated except that fetal mouse or adultrat hepatocytes were added to the sodium alginate solution in amounts of10⁵ hepatocytes/ml of alginate solution and the distance from the tip ofthe needle to the surface of the calcium chloride solution was decreasedto 7 mm. The resulting microcapsules were spherical in appearance andhad a diameter of 250 μm (±50 SD). The presence of viable hepatocyteswas demonstrated by Trypan Blue staining and histology, even after morethan 4 weeks in culture at 37° C.

Example 6

This Example illustrates the effect of needle parameters on gel dropletsize.

The procedure of Example 1 was repeated, except that a 26 gauge needlehaving a 22-degree bevel was used in place of the 22 gauge needle havingthe 90-degree bevel. The resultant gel droplets had a diameter of 170 μm(±30 SD), demonstrating the smaller diameter gel droplets andconsequently microcapsules can be formed by using a smaller diameterneedle.

SUMMARY OF DISCLOSURE

In summary of this disclosure, the present invention provides a noveldroplet generation procedure using electrostatic forces which isparticularly useful in the microencapsulation of living tissue or cellsto form small diameter microcapsules suitable for cardiovascularinjection. Modifications are possible within the scope of the invention.

What we claim is:
 1. A method of forming spherical, smooth and uniformmicroapsules containing living tissue or cells, which comprises:forminga suspension of living tissue or cells in a physiologically-compatiblemedium containing a water-soluble substance which can be reversiblygelled to provide a temporary protective environment for the livingtissue or cells, extruding said suspension downwardly from a source at afirst location comprising an axially downwardly directedelectroconductive needle, charging the extruded material with a chargeof one polarity, establishing a charge of opposite polarity at a secondlocation spaced vertically below said first location, providing adifference in voltage between the first location and second locationcyclically in pulses of a magnitude from about 1 to about 25 kv at afrequency of about 10 to about 100 sec-1 for a pulse duration of about 1to about 6 m. sec. to effect continuous production of droplets from theextruded material and to draw the droplets so-formed towards said secondlocation, collecting said droplets in a recipient medium which is ahardening solution which reacts with said water-soluble substance andform discrete, spherical microcapsules from each of said droplets ofdiameter less than about 700 microns, and subsequently forming apermanent biocompatible semi-permeable membrane having anegatively-charged outer surface about each of said microcapsules whichforms a core of the resulting spherical, smooth and uniformmicrocapsules, said membrane permitting nutrients and oxygen to flow tothe core material and metabolic products to flow therefrom whileretaining the core material within the microcapsule, said membranecomprising ionically-interacted biocompatible materials and having athickness about 4 to about 6 microns, said resulting microcapsules beingsuitable for cardiovascular injection.
 2. The method of claim 1 whereinsaid recipient medium is provided at said second location.
 3. The methodof claim 1 when said second location comprises a ring ofelectroconductive material surrounding the axis of said needle and beingconcentric to said axis and such recipient medium is location below saidlocation.
 4. The method of claim 1 wherein said water-soluble substanceis a gellable polysaccharide gum.
 5. The method of claim 4 wherein saidpolysaccharide-gum is sodium alginate.
 6. The method of claim 1 whereinsaid hardening solution comprises an aqueous calcium chloride solution.7. The method of claim 6 wherein said semi-permeable membrane is formedabout each of the microcapsules by ionic reaction between free acidgroups in the surface layer of the gelled gum and biocompatible polymerscontaining acid-reactive groups.
 8. The method of claim 7 wherein saidbiocompatible polymer is a polyamino acid.
 9. The method of claim 8wherein said polyamino acid is polylysine having a molecular weight ofabout 10,000 to about 30,000.
 10. The method of claim 9 wherein saidpolylysine is reacted with the microcapsules for at least six minutes.11. The method of claim 7 where said biocompatible polymers, afterreaction with the gelled gum, is treated with a non-toxic biocompatiblewater-soluble polymeric materials capable of ionic reaction with freeamino groups to form an outer negatively-charged coating about themembrane.
 12. The method of claim 11 wherein said water-soluble polymermaterial is a polysaccharide gum.
 13. Themethod of claim 12 wherein saidpolysaccharide gum is sodium alginate.
 14. The method of claim 1including reliquifying the core material.
 15. The method of claim 1wherein said living tissue is islets of Langerhans, whereby saidcardiovascular injection permits control of blood sugar levels in ananimal body.
 16. The method of claim 1 wherein said microcapsules have adiameter of about 150 to about 500 microns.